Biofuels derived from microalgal lipids have demonstrated a promising potential as future renewable bioenergy. However, the production costs for microalgae-based biofuels are not economically competitive, and one strategy to overcome this limitation is to develop better-performing microalgal strains that have faster growth and higher lipid content through genetic screening and metabolic engineering. In this work, we present

Botryococcus braunii has long been known as a prodigious producer of liquid hydrocarbon oils that can be converted into combustion engine fuels. This draft genome for the B race of B. braunii will allow researchers to unravel important hydrocarbon biosynthetic pathways and identify possible regulatory networks controlling this unusual metabolism.

Plants react to biotic and abiotic stresses with a variety of responses including the production of reactive oxygen species (ROS), which may result in programmed cell death (PCD). The mechanisms underlying ROS production and PCD have not been well studied in microalgae. Here, we analyzed ROS accumulation, biomass accumulation, and hydrocarbon production in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO3, salicylic acid (SA), methyl jasmonate, and acetic acid. We also identified and cloned a single cDNA for the B. braunii ortholog of the Arabidopsis gene defender against cell death 1 (DAD1), a gene that is directly involved in PCD regulation. The function of B. braunii DAD1 was assessed by a complementation assay of the yeast knockout line of the DAD1 ortholog, oligosaccharyl transferase 2. Additionally, we found that DAD1 transcription was induced in response to SA at short times. These results suggest that B. braunii responds to stresses by mechanisms similar to those in land plants and other  organisms.

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We analyzed the reactive oxygen species (ROS) accumulation in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO3, salicylic acid (SA), methyl jasmonate, and acetic acid. A staining assay using the fluorescent dye CellROX Green was used. CellROX Green is a fluorogenic probe used for measuring oxidative stress in live cells. The dye is weakly fluorescent inside cells in a reduced state but exhibits bright green photostable fluorescence upon oxidation by ROS and subsequent binding to DNA. The large amount of liquid hydrocarbons produced and excreted by B. braunii, creates a highly hydrophobic extracellular environment that makes difficult to study short times defense responses on this microalga. The procedure developed here allowed us to detect ROS in this microalga even within a short period of time (in minutes) after treatment of cells with different stress inducers.

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Botryococcus braunii produce liquid hydrocarbons able to be processed into combustion engine fuels. Depending on the growing conditions, the cell doubling time can be up to 6 days or more, which is a slow growth rate in comparison with other microalgae. Few studies have analyzed the cell cycle of B. braunii. We did a bioinformatic comparison between the protein sequences for retinoblastoma and cyclin-dependent kinases from the A (Yamanaka) and B (Showa) races, with those sequences from other algae and Arabidopsis thaliana. Differences in the number of cyclin-dependent kinases and potential retinoblastoma phosphorylation sites between the A and B races were found. Some cyclin-dependent kinases from both races seemed to be phylogenetically more similar to A. thaliana than to other microalgae. Microscopic observations were done using several staining procedures. Race A colonies, but not race B, showed some multinucleated cells without chlorophyll. An active mitochondrial net was detected in those multinucleated cells, as well as being defined in polyphosphate bodies. These observations suggest differences in the cell division processes between the A and B races of B. braunii.

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